This invention concerns methods and apparatus for imaging, particularly the imaging of fluorescing samples of the type in which the sample is first illuminated with an excitation radiation such as ultra-violet light and is subsequently interrogated for any resulting emission light due to fluorescence from within the sample.
Typically a sample will comprise an array of a large number of sites each containing a small quantity of material or mixture of materials under investigation, and one convenient array comprises a plate containing a large number of wells arranged in a rectangular matrix each of which comprises a site; or a membrane, or a dish, over the surface of which exist a large number of reaction sites which are to be investigated.
For the purpose of investigation an image of each of the sites (preferably all at the same time) is formed at the input to a detector which converts light into electrical signals. The detector may comprise a CCD camera which may be cryogenically cooled or may comprise a CCD camera preceded by or including an image intensifying device.
It is known from U.S. Pat. No. 4,922,092 to utilise a fibre optic device to couple the light emitted from such an array of sites to such imaging devices.
It is an object of the present invention to provide a device by which an array of such sites can be coupled to a source of excitation radiation (such as a source of UV light), as well as to the input of an imaging device, so that after exposure to excitation radiation, the sites can be inspected for any fluorescence arising therefrom.
The invention will be of particular benefit where the sites are small and closely packed together as in the case of high density multi-well plates.
According to one aspect of the present invention there is provided a fibre optic coupling plate having a sample viewing face for receiving light from a sample, an output window for conveying sample originating (emitted) light to an imaging detector and an additional window through which excitation radiation can be projected, wherein there are provided a primary light path through the plate made up of optical fibres which will convey light entering the viewing face, directly and with minimal loss, to the output window, and a secondary light path separate from the primary light path, by which excitation radiation entering the additional window is conveyed to the viewing face for irradiating the sample.
The coupling plate preferably includes radiation reflecting or absorbing means to restrict the exit of excitation radiation therefrom except as required to irradiate the sample.
To this end, and where the material forming the coupling plate is not opaque to the excitation radiation, the optical fibres forming the primary path may be coated or sleeved with a material resistant to the transmission of the excitation radiation, and those areas of the output window not occupied by the optical fibre ends are rendered impervious to excitation radiation to prevent the latter from exiting in the direction of the detector.
The primary light path fibres are preferably formed from a material which readily transmits light emitted from the sample but only poorly transmits excitation radiation wavelengths.
The secondary path may comprise a second set of optical fibres which are bundled with the first set of fibres forming the primary path, and the fibres of both sets terminating in and comprising at least part of the sample viewing face.
The ends of the fibres from the two sets may be interspersed in a random manner across the face of the sample viewing window or may be arranged in a particular pattern so that the fibres of the first set bear a fixed relationship both in position and number relative to the fibres of the second set.
Where the second path is comprised of fibres, they are preferably formed from a material which readily transmits excitation radiation wavelengths.
In a preferred arrangement, the secondary path comprises selected optical material which is capable of transmitting the excitation radiation and which surrounds, but is optically screened, from the optical fibres which convey the image light to the output window.
The selected material is preferably in the form of a sleeve around each of the fibres of the first set, although in some circumstances it may be in the form of a block of material through which all the fibres in the first set extend.
In a particularly preferred arrangement, the coupling plate is formed from the said selected optical material and the fibres conveying the light from the sample to the detector are coated or sleeved with a material which is impervious at least to the excitation radiation wavelengths.
The optical screening is most simply provided by a coating the fibres making up the said first set. In known manner the coating material is selected so that it not only serves to reflect any internal radiation (so as to improve the efficiency of the fibres), but is also selected so as to be reflective at the wavelengths of the excitation radiation so that it not only prevents the latter from entering the fibres but also renders available and useful reflected excitation radiation for subsequent reflection and possible emission out of the coupling plate towards the sample as required.
By coating the fibres with a suitable material light within the fibres is completely prevented from escaping into the plate material surrounding them.
According to a particularly preferred aspect of the invention, each of the optical fibres in the first set is replaced by a bundle of parallel very small diameter fibres, each of which is coated as aforesaid to prevent the ingress of excitation radiation and the exit of light from within the fibre.
Preferably the small diameter fibres in each bundle are also separated one from the other by the said selected optical material which also surrounds and separates one bundle of fibres from adjacent bundles of fibres.
In one embodiment the plate includes a large number of separate fibres, (or fibre bundles) as aforesaid, distributed substantially uniformly across at least the input face area of the plate, each fibre or bundle of fibres extending from the input face to another face thereof, which serves as the output window.
If the density (ie the number per unit area) of the fibres (or bundles), across the face, is sufficiently high, it may not be essential for the fibres or bundles to be arranged in any particular pattern or have any particular spacing as between one and the next so long as each is a discrete isolated light path.
According to a further aspect of the invention, the input face area may be larger than the input window area so that the density of the fibres or bundles in the latter is greater than is the density of the fibres or bundles in the former.
According to a further aspect of the invention, where a coupling plate as aforesaid is to be used with a sample having a regular array of reaction sites, (or wells) then the fibres (or bundles of fibres) are preferably arranged (at least over the input face of the plate) in the same pattern and with the same interstitial spacing as are the sites in the sample, so that registration is possible between sites and fibres (or fibre bundles), one fibre (or fibre bundle) registering with each site.
Where the coupling plate is formed from selected optical material and the latter is used as the medium for conveying the excitation radiation, it is simply necessary for part of the well or site to communicate with a small region of the material forming the coupling plate to enable excitation radiation to exit thereinto.
Where each site is xe2x80x9cseenxe2x80x9d by one or a few large area fibres, the area of the end of each such fibre (or group of fibres) in the input face must be somewhat less than the area of the site or well with which it cooperates, so that at least part of each site registers with an area of the input face of the coupling plate not occupied by optical fibre(s) leading to the output window, so that excitation radiation can exit therethrough into the site or well.
Where for example the wells are circular and large diameter circular section fibres are centred relative to the wells or sites, the diameter of the fibre being less than that of the well, there will be an annulus of selected optical (plate) material around the fibre through which excitation radiation can pass to the sample.
However, the more preferred arrangement comprises a fibre bundle associated with each site, in which the individual fibres are spaced apart by the said selected optical material from which the plate is also formed, and the area of the bundle is commensurate with the area of the site or well. Excitation radiation is conveyed to the site or well through the material separating the individual fibres in each bundle. This has the benefit that the excitation radiation will tend to be more or less uniformly distributed over the area of the bundle and therefore the site or well so that excitation radiation is very closely coupled to all regions in the well or site which might react thereto. Where some assays are concerned, this is of considerable importance.
In a coupling plate as aforesaid, it has been found that excitation radiation can be conveyed to the sites or wells, coupled to the input face thereof, by simply directing excitation radiation edgewise into the plate when and as required. Where the sites and fibres (or fibre bundles) are arranged in the same pattern and with the same spacing, the transfer of excitation radiation into the sites is best accomplished when the fibres (or bundles) are centred relative to the sites.
In one arrangement the number of fibres (or fibre bundles) is the same as the number of sites in the sample. However with the tendency towards ever increasing numbers of sites per sample, an alternative arrangement is one in which the number of fibres (or fibre bundles) is a fraction of the number of sites in the sample and all of the latter can be inspected, albeit in a sequence of steps, by moving the sample relative to the coupling plate input face (or vice versa) until all of the sites have been brought into registry with the fibres (bundles of fibres) at one time or another in the said sequence of steps.
Where the excitation radiation is in the ultra-violet the said select optical material may be silica glass.
Where the fibres extend into the input window surface, the area of the window surrounding each fibre (whether large individual ones or small diameter fibres making up bundles) may be rendered opaque to ultra-violet radiation by acid etching the silica glass, coating the whole surface of the output window with a titanium dioxide or epoxy and then grinding the coating material down to expose the ends of the fibres while leaving the coating material in the troughs created by the etching process in the surface of the glass surrounding the fibres. In this way only the fibre ends extend through the coating material and by a choice of suitable coating material such as suggested, ultra-violet radiation within the coupling plate can be prevented from exiting around the fibre ends towards the detector.
As indicated above, the excitation radiation may be projected edgewise into the coupling plate and this edgewise projection may take one of a number of different forms.
In one arrangement a single source of excitation radiation is directed through a single window in the edge of the plate, and the edge and output face surface of the plate are all coated (eg with titanium dioxide/epoxy) so that the only exit path for excitation radiation is towards the sample, through the optical material from which the coupling plate is formed which surrounds the fibres in the input face.
Modifications of this arrangement involve the use of two, three or more such radiation sources arranged symmetrically around the coupling plate, each having its own input window through which radiation therefrom can be projected into the coupling plate.
In a preferred arrangement, the whole of the edge of the plate is left as an input window for excitation radiation, and the coupling plate is embraced and completely surrounded by excitation radiation from one or more sources arranged thereon around. Reflecting means may be provided external to the plate more evenly to distribute the radiation towards the edge of the coupling plate.
Filters or shutters may be provided so as to prevent the ingress of excitation radiation except when required. Absorption filters may be provided for absorbing any excitation radiation which may leak via the fibres at the output window. This latter feature may be important where the detector is sensitive to the excitation radiation wavelengths as well as the fluorescence wavelengths (as is normally the case) since any leakage of excitation radiation into the input ends of the fibres either, directly, or after reflection from the sample, could swamp or even destroy the detector the sensitivity certainly temporarily, if not permanently.
By coupling the plate directly to the sites or wells and to the input window of the detector means (whether a CCD camera or an image intensified camera or an image intensifier), cross-talk (caused by a region of the detector receiving light from the fluorescence occurring in two more sites) has been found to be virtually eliminated. This allows adjacent sites to be imaged simultaneously which speeds up the inspection process.
This advantage is also important since it may be desirable to be able to irradiate and then check for fluorescence very quickly and without previous multiple exposures to excitation radiation. The invention allows all of this to occur since all of the sites which are irradiated are immediately available for detection, and in general sites which are not available for detection are not irradiated.
The invention also lies in a method of checking for fluorescence in a sample, comprising the steps of irradiating the sample with excitation radiation, removing or ceasing the excitation radiation, and conveying any fluorescent light arising from the sample via fibre optic means to the input of a detector, wherein the excitation radiation is projected into a coupling plate through which the optical fibres extend, and the plate includes an optical path by which the excitation radiation can exit to the sample.
The invention also lies in apparatus for imaging fluorescence light arising from reaction sites in a pre-excited sample, comprising a sample holder, a coupling plate adjacent the holder having fibre optic means extending from the face thereof adjacent the sample holder to an input window of a detector such as an image intensifying CCD camera, a source of excitation radiation, housing means containing the source and the coupling plate, an optical path through the coupling plate for conveying excitation radiation to at least one area of the face of the plate adjacent the sample holder, means preventing excitation radiation from leaving the coupling plate at least in the direction of input window of the detector, means for controlling either the excitation radiation source or shutter means, so as to enable excitation radiation to be projected into the coupling plate only when required, signal processing means receptive of electrical signals from the detector means for at least storing and/or displaying the electrical signals as visually distinguishable regions in a visual display corresponding to part of or the whole of the input window of the detector. To this end the signal processing means may comprise windowing and/or scaling means to enable signals from only part of the area of the input window to be displayed as a full screen display in the visual display device.
The latter may comprise a television camera or LCD panel and preferably is such as to enable different colours to be displayed.
Preferably the signal processing circuit means attributes different colours to different intensities of light incident on the detector.
Where wavelength discrimination is also required, filter means may be provided to enable the detector to be responsive first of all to one wavelength, and then another.
Each image may be stored and then displayed either in sequence or simultaneously in registration. The visually distinguishable regions therein corresponding to the different light emissions may be displayed in different colours for example according to wavelength and/or intensity of the light received by the detector.
In some circumstances, optimal coupling between the sample and the plate and/or between the plate and detector has been found to occur when a very small gap is provided between the sample and the input face of the coupling plate and/or between the output window and the input window of the detector. The size of any such gap must be such as to not permit an unacceptable increase in cross-talk to occur. However a gap between the sample and the input face of the coupling plate, assists in permitting sliding movement of one device relative to the other, where as will normally be the case, the area of the input face of the coupling plate is significantly smaller than the overall area of the sample, so that it is necessary to move the sample relative to the coupling plate (or vice versa) to allow all of the sites on the sample to be irradiated and inspected.
In apparatus as aforesaid, drive means may be provided for accurately indexing the sample holder relative to the coupling plate (or vice versa).
The drive means is preferably computer controlled and the latter may also be programmed to control the production of the excitation radiation (or the operation of a shutter or other device for exposing the coupling plate to the excitation radiation), as required, and for whatever duration is required.
Both the indexing and the exposure and/or the exposure duration are preferably separately and independently programmable.
Although the invention is of particular application to multi-well sample plates in which each of the wells comprises a reaction site which is to be exposed and investigated, and these are essentially viewed from below through the coupling plate, the invention is not limited to such an arrangement, and where the sites are located on a medium or support surface through which light is only inefficiently or inadequately transmitted, the coupling plate may be inverted so that it is in contact with, or very close to, the surface on which the sites are located, so that excitation radiation is now directed in a downward sense through the coupling plate and it is upwardly directed light caused by fluorescence instigated by the irradiation which is detected.
Where universal apparatus is to be provided in which a sample can be inserted for viewing either above or below, two coupling plates may be provided with appropriate light paths from a common excitation radiation source, or separate excitation radiation sources may be provided, one for each coupling plate. The one plate may be above, and the other below, a sample holder which is adapted to receive multi-well plates or petri dishes or the like and which can be viewed from above or below. The fibre optic bundle in the upper coupling plate may be extended beyond the plate in the form of an umbilical which is curved through 180xc2x0 to extend in a downward sense to an output window remote from the upper coupling plate. The two output windows, the one relating to the lower coupling plate and the other corresponding to the exit end of the fibre optic bundle from the other coupling plate, may be arranged side by side in the same plane and a single detector may be movable by drive means from a position where it registers with the output window of the underside plate to a position where it registers with the exit end of the fibre optic bundle from the upper plate.
Alternatively prism means, mirror means or lens means may be used to enable light from both fibre optic bundles to be imaged onto the input window of a single detector, although because of aperture problems associated with lenses this latter option may not normally be available in low light conditions.
Alternatively two separate detectors are provided one for the upper and one for the lower coupling plates.
Drive means may also be provided within such apparatus for moving the coupling plate (5) towards and away from the sample holder, and where two such coupling plates are provided, separate drive means is provided for each, so that having inserted a sample and indicated to the apparatus whether it is to be inspected from above or below, the appropriate drive means is engaged for moving the appropriate coupling plate towards the sample. Sensing means may be provided for determining when the coupling plate occupies the desired position for irradiating and inspecting the sample.
In the event that radiation and inspection is required from above and below, both coupling plates may be moved into position to permit irradiation and detection of fluorescence by one and/or then the other or both simultaneously.
It will be noted that in the case of an apparatus having above and below coupling plates, the sample may be irradiated using the excitation radiation source associated with one coupling plate and any resulting fluorescence may be detected using the detector associated with the other coupling plate.
According to a further feature of the present invention the optical fibres forming the primary light path in the coupling plate are arranged in groups, and each group is arranged to receive emission radiation from a single sample site, to transfer the emission radiation from that site to a discrete region of the output window viewed by the camera.
Conveniently the groups form fibre optic bundles.
Preferably the external surface of the fibres making up each bundle, or at least the outside surface of the bundle, is formed with an opaque coating, typically a radiation reflecting surface. Conveniently titanium dioxide paint may be used.
Typically the area of cross-section of the input end of each bundle is commensurate with but a little smaller than the cross-section area of the sample site from which radiation may be emitted into the bundle.
Since it is necessary for excitation radiation to enter the sample site so as to initiate fluorescence and thereby generate emission radiation, the input end of each bundle is preferably spaced from the underside of the reaction site by a small distance and an annular window is provided through which excitation radiation can pass into the base of the reaction site, around the end of the bundle.
Preferably an annular shield is provided around the end of the bundle so as to prevent direct ingress of excitation radiation into the input end of the bundle, and to generally direct excitation radiation towards the outer annular region of the reaction site, through which region the excitation radiation passes.
In a preferred arrangement, a plurality of reaction sites are arranged in the form of wells in a so-called multi-well plate, the wells and the fibre optic bundles are generally circular in cross-section, the well sites all have the same diameter, the fibre optic bundles are all of another slightly smaller diameter, and the circular cross-section fibre optic bundles are centrally aligned, one below each well site. For simplicity the base of each well is transparent to both emission and excitation radiation and in order to reduce cross-talk between adjacent well sites an apertured opaque plate may be located immediately below the wells, the apertures in the plate being circular and spaced so as to align with the wells. By making the diameter of each aperture just a little larger than the internal diameter of the corresponding well with which it is aligned, and by making the outside diameter of the fibre optic bundle viewing the underside of the well slightly smaller in diameter than the internal diameter of the well, a small annular region will exist through which excitation light can enter the well, around the outside of the fibre optic bundle and within the opening in the opaque plate.
Instead of forming openings in an opaque plate, the latter may be formed from material which is transparent to both excitation and emitted wavelength, and those regions which are required to be opaque are coated or impregnated with an appropriate non transmittive material, typically a reflective material, so as to inhibit the passage of either excitation or emitted radiation except through the untreated window regions left in the plate.
The windows may be formed from the same material as is the base of the wells. A clear polystyrene material may be used.
Where the underside of the well window is spaced from the end of the fibre optic bundle the gap between it and the end of the fibre optic bundle is preferably filled with a transparent material having a similar refractive index to that of both the well base and, if provided, the intermediate window.
Where clear polystyrene is used for the well base and the window material, a clear polystyrene plug may be located between the end of the fibre optic bundle and the underside of the base or the window in the plate.
Where an opaque and preferably reflecting annular shield is provided around the end of the fibre optic bundle to reduce the possibility of excitation radiation directly entering the fibres in the bundle, the annular shield preferably also extends around the plug of transparent material between the end of the fibre optic bundle and the underside of the well.
In a particularly preferred arrangement, the end of the fibre optic bundle may be surrounded by an annular sleeve of excitation wavelength transmittive material, the inner surface of which is formed with an opaque and possibly reflective coating, to prevent the passage of excitation or emitted radiation therethrough, and the fibre optic bundle terminates part way through the sleeve and a transparent plug may be fitted into the remaining space within the annular sleeve.
The annular sleeve may serve as a diffuser and be constructed accordingly.
Typically the external diameter of the annular sleeve is the same as the diameter of the window in the opaque plate.
Where the opaque plate is apertured, the annular sleeve may be fitted and secured into the window so that one end of the annular sleeve, together with the central plug above the fibre optic bundle end, is flush with the surface of the apertured plate which cooperates with the underside of the well(s).
The apertured plate may be bonded to the underside of the wells or a small gap may exist as would be the case where it is necessary to be able to replace one array of wells with another.
Typically the wells are formed in a single plate called a well-plate.
According to a preferred feature, the transparent plug fitted within the annular sleeve surrounding the end of the fibre optic bundle may be formed from filter material so that radiation of one wavelength is favoured relative to radiation of another.
By selecting the characteristics of the filter material forming the plug, the latter may serve to restrict the entry of excitation wavelengths into the fibre optic bundle and/or favour emitted radiation of one wavelength as opposed to emitted radiation of other wavelengths.
In one embodiment of the present invention, the output end of the fibre optic plate is coupled to a camera input window via a filter. The purpose of the filter is to restrict wavelengths entering the camera to those of expected, or wanted, emissions so as to ideally remove from the camera input any radiation at unwanted wavelengths such as stray excitation radiation transmitted via the fibre optic plate, or the like.
In an example involving 96 fibre optic bundles, the 96 outputs of the bundles may be arranged in any convenient configuration or aspect ratio depending on the output of the camera to which the image is to be applied. Where the latter is generally circular, the 96 rods may be arranged in a generally circular or hexagonal array so as to substantially fill the entrance window of the camera, and if the latter is 40 mm diameter and the rods fill an area of 32xc3x9742 mm2, there should be adequate spacing between rod centres (approximately 2.5 mm) to ensure minimal cross-talk between bundles. Where an emission filter is inserted between the plate and the camera input, this should be as thin as possible, and may need to be less than 0.5 mm to ensure acceptable levels of cross-talk.